Project Summary/Abstract The human intestines are colonized by trillions of microorganisms, termed the gut microbiota, which are thought to rival the number of our own cells. Together, these microbes metabolize small molecules within the intestinal lumen through the activities of bacterial enzymes that carry out biochemical transformations. Growing evidence suggests that these small-molecule metabolites confer major benefits to host immunity and physiology. However, the enzymes and biochemical pathways that produce these molecules remain poorly understood. This proposal seeks to develop chemical approaches to understand the metabolic activity of the gut microbiome to better understand metabolite production in the gut and how it contributes to health and disease. The overarching hypothesis guiding this work is that activity-based profiling can be used to identify active bile salt hydrolases (BSHs) within the gut microbiome, which produce bacterially-modified bile acids that have important functions in physiology and disease. We will address this hypothesis with the following studies: Develop selective chemical probes for labeling active bile salt hydrolases. Building on our strong preliminary data based on a novel activity-based probe that can label active BSH, we will develop improved probes that exhibit greater selectivity and specificity for different isoforms of this critical enzyme that have different substrate preferences and are produced by various strains of bacteria. This panel of chemical probes will enable a greater understanding of BSH activities from diverse bacterial strains within the gut microbiome. Profile active bile salt hydrolases from mouse and human gut microbiomes in health and disease. Building on preliminary data demonstrating changes in BSH activity in colitis, which is associated with dysbiosis, we will apply our panel of optimized probes to mouse and human gut microbiomes to profile active BSHs in health and disease, using both mouse models of colitis and human patient samples. These results will inform on changes in BSH activity during health and inflammatory diseases that are influenced by the gut microbiome. Visualize bile salt hydrolase activity in mouse and human intestines in health and disease. We will apply the chemical probes to image active BSH within the intestinal tissue from mice and humans in both health and disease. These studies will determine the localizations of gut bacterial niches that are actively metabolizing bile acids during health and inflammatory diseases that are affected by gut microbiome dysbiosis, e.g., colitis. Current technologies based on metagenomics are limited in their ability to report on genes that are present within the microbiome. Our chemical approach will define how activities of enzymes within the gut microbiome carry out metabolism of important small-molecule metabolites that regulate host physiology and pathology. Broadly, our tools will contribute to a deeper understanding of host-microbiome interactions in the gut and how this relationship influences human health and disease.